CAJ | 학술논문

目的探讨非转移性黑素瘤糖蛋白B(GPNMB)对黑素瘤细胞增殖、迁移及黑素形成的影响.方法以人原代黑素细胞、黑素瘤细胞系M14、G-361为研究对象,采用免疫荧光法检测GPNMB表达量.细胞分为三组,实验组加入GPNMB-siRNA,阴性对照组加入阴性对照-siRNA,空白对照组为未经处理的细胞.实时定量逆转录(RT)-PCR检测GPNMB-siRNA的转染效率,以噻唑蓝法和Transwell小室法分别检测细胞的增殖及侵袭能力,并用分光光度计检测黑素水平,结果进行t检验分析.结果 GPNMB-siRNA转染下调了GPNMB mRNA及蛋白的表达水平,并显著抑制黑素瘤细胞的增殖和迁移能力.其中黑素瘤M14细胞和G361细胞的增殖能力分别降低35％和40％,迁移能力分别降低49％和51％；同时,黑素细胞、黑素瘤M14细胞和G361细胞黑素形成分别降低73％、82％和69％.结论 GPNMB缺失能抑制黑素瘤的增殖、迁移以及黑素瘤中黑素的形成,表明GPNMB在黑素瘤的发生发展中起重要作用.
목적 탐토비전이성흑소류당단백B(GPNMB)대흑소류세포증식、천이급흑소형성적영향.방법 이인원대흑소세포、흑소류세포계M14、G-361위연구대상,채용면역형광법검측GPNMB표체량.세포분위삼조,실험조가입GPNMB-siRNA,음성대조조가입음성대조-siRNA,공백대조조위미경처리적세포.실시정량역전록(RT)-PCR검측GPNMB-siRNA적전염효솔,이새서람법화Transwell소실법분별검측세포적증식급침습능력,병용분광광도계검측흑소수평,결과진행t검험분석.결과 GPNMB-siRNA전염하조료GPNMB mRNA급단백적표체수평,병현저억제흑소류세포적증식화천이능력.기중흑소류M14세포화G361세포적증식능력분별강저35％화40％,천이능력분별강저49％화51％；동시,흑소세포、흑소류M14세포화G361세포흑소형성분별강저73％、82％화69％.결론 GPNMB결실능억제흑소류적증식、천이이급흑소류중흑소적형성,표명GPNMB재흑소류적발생발전중기중요작용.
Objective To estimate the effect of glycoprotein (transmembrane) nonmetastatic melanoma protein B (GPNMB) on the proliferation and migration of as well as melanogenesis in melanoma cells.Methods The expression of GPNMB was detected by immunofluorescence assay in two melanoma cell lines M14 and G-361,as well as in primary human melanocytes.Then,the three kinds of cells each were classified into three groups:experimental group treated with small interfering RNA targeting GPNMB (GPNMB-siRNA),negative control group treated with the negative control siRNA,blank control group remaining untreated.Methyl thiazolyl tetrazolium (MTT) assay,transwell invasion assay and spectrophotometry were performed to evaluate cell proliferation activity,invasion potential and melanin levels,respectively.Statistical analysis was done using Student's t test.Results GPNMB was expressed in both melanoma cells and melanocytes.The transfection with GPNMB-siRNA down-regulated the mRNA and protein expressions of GPNMB in,and markedly suppressed the proliferation and migration of,melanoma cells.In detail,the proliferative activity (expressed as the absorbence value at 570 nm) of M14 and G361 cells was reduced by 35％ and 40％ respectively,the migration activity of M14 and G361 cells by 49％ and 51％ respectively,and the melanin levels in melanocytes,M14 cells and G361 cells by 73％,82％ and 69％ respectively,in the experiment group compared with those in the blank control group.Conclusions The siRNA-mediated silencing of GPNMB could effectively inhibit the proliferation of,invasion of and melanogenesis in melanoma cells,which suggests that GPNMB plays critical roles in the initiation and progression of melanoma.